P449: NOVEL, FIRST-IN-CLASS, SOMATIC IDH2 DELETION-INSERTION VARIANT C.516_518DELINSTGC CONFERS BORDERLINE PHENOTYPE AND DOES NOT SHOW SUSCEPTIBILITY TO ENASIDENIB IN-VITRO
Authors: L. Ruhnke, D. M. Poitz, S. Herold, S. Dressler, C. Dill, M. Peitzsch, U. Oelschlägel, J. Frimmel, D. Kunadt, T. Kretschmann, H. Altmann, F. Stölzel, C. Röllig, T. Chavakis, G. Baretton, K. Schäfer-Eckart, M. Bornhäuser
Journal: HemaSphere (2022)

Background: Mutations in IDH2 are among the most common genetic alterations in patients with acute myeloid leukemia (AML). So far, only substitution variants (also known as single nucleotide variants (SNVs)) - in particular affecting the hotspots at codon Arg140 and Arg172 - have been identified. . Aims: Here we report and functionally characterize a novel, first-in-class, somatic IDH2 deletion-insertion (delins) variant affecting codons Arg172/His173 identified in a patient diagnosed with AML with myelodysplasia related changes (AML-MRC). Methods: The novel delins variant was identified performing amplicon-based targeted next generation sequencing (NGS), Sanger sequencing was used for verification. For functional evaluation high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS), global DNA methylation and DNA hydroxymethylation assays and in-vitro drug testing was performed; AML patients without evidence of IDH1 or IDH2 mutations and patients with IDH2Arg172 mutation served as negative and positive controls, respectively. Results: A 63-year-old male patient (unique patient number (UPN) 1) was diagnosed with AML-MRC. Further work-up revealed an aberrant karyotype (47,XY,+11/ 46,XY,der(7)t(7;11)(q31;q12)/ 46,XY), a partial tandem duplication of KMT2A (KMT2A-PTD) as well as variants in IDH2, DNMT3A and NRAS (Figure 1.) (no CBFB::MYH11, PML::RARA or RUNX1::RUNX1T1 fusion, no ASXL1, CEBPA, FLT3, NPM1, RUNX1 or TP53 mutation) (Figure 1A). He received induction therapy with liposomal daunorubicin/cytarabine, achieved CR and underwent allogeneic hematopoietic cell transplantation. Of note, the IDH2 variant identified was a novel three base-pair deletion-insertion variant (c.516_518delinsTGC) leading to the replacement of amino acids arginine and histidine at codon 172 and 173 for serin and alanine (p.Arg172_His173delinsSerAla), variant allel frequency was 44%. The novel delins variant could be validated performing Sanger sequencing; parallel Sanger sequencing of buccal saliva DNA ruled out germline origin (Figure 1B). Since there are no valid in-silico prediction tools for delins variants, we decided to assess the potential pathogenicity of the novel alteration. First, we applied LC-MS/MS to evaluate 2-hydroxyglutarate (2-HG) levels as compared to BM-MNCs obtained from IDH1/IDH2WT AML patients (UNP2-4) and IDH2MUT AML patients (UPN5-10). In line with previous reports, 2-HG levels were increased in patients with IDH2 mutations as compared to their IDH1/IDH2WT counterparts (1000-fold). However, in UPN1 only slightly elevated 2-HG levels were found (10-fold) (Figure 1C); no differences in isocitrate or ketoglutarate levels were seen (data not shown). Similar results were obtained when performing DNA methylation and DNA hydroxymethylation studies. While the percentage of methylated DNA in UPN1 was comparable to the amount of 5-methylcytosine (5-mC) seen in the IDH2 mutated group (UNP5-10), 5-hydroxymethylcytosine (5-hmC) levels in UPN1 tended towards those observed in the IDH2WT cohort (UPN2-4) (data not shown). Finally, we performed in-vitro drug testing to evaluate response to the IDH2 inhibitor (IDH2i) enasidenib. Interestingly, enasidenib did not affect granulocytic differentiation of BM-MNCs as assessed by CD11b, CD15 and CD16 flow cytometry (Figure 1D). Image:Summary/Conclusion: Here, we report a novel, somatic delins variant in IDH2, which, albeit affecting the known Arg172 hotspot, does not show susceptibility to the IDH2i enasidenib in-vitro. Our report argues for functional characterisation of novel variants to ensure a valid biomarker-driven therapy in AML patients.